Articles in the Category ”Poster Abstracts”
The Digested Guide to ASMS 2019
Many of Genovis’ customers have submitted abstracts for ASMS 2019 in Atlanta, in which the SmartEnzymes have been used. Below is a digested guide to these abstracts so that you can plan your days at ASMS.
To read the full poster abstracts, visit the Online Planner for ASMS, and paste the abstract titel in the search field.
Sunday, June 2
New Enzymatic Workflows for Analysis of O-Glycosylated Biopharmaceuticals
Andreas Nägeli
Thermo Fisher Scientific User Meeting
Pharma/BioPharma Breakout Session 9:30 AM – 12.15 PM
Marquis Ballroom C
Monday, June 3
Improved middle-down characterization of antibodies using multiple ion activation techniques and Proton Transfer Reaction on a modified Orbitrap mass spectrometer
Romain Huguet
MP 676, Poster Session 10.30 AM – 2.30 PM
Direct Determination of Antibody Chain Pairing by Top-Down Mass Spectrometry using Electron Capture Dissociation and Ultraviolet Photodissociation
Jared Shaw
Room B302-305, Oral Presentation 3.50 PM – 4.10 PM
Unraveling a complex immunoprotein profile in multiple myeloma with middle-down de novo sequencing and native mass spectrometry
Valerie J Winton
Room A411-412, Oral Presentation 9.30 AM – 9.50 AM
Middle Down Approach for the Characterization of Monoclonal Antibodies After Ides Digestion and ETD Fragmentation
Colin Wynne
MP 782, Poster Session 10.30 AM – 2.30 PM
Tuesday, June 4
Analysis of Zika Viral Polyprotein N- and O-glycosylation Using a Novel Lectin-chemoenzymatic Enrichment
Shuang Yang
TP 655, Poster Session 10.30 AM – 2.30 PM
OpeRATOR® and GlycOCATCH™
Comprehensive Characterization of Antibody Drug Conjugates Enabled by Top-down and Middle-down Mass Spectrometry Strategies
Eli J Larson
TP 601, Poster Session 10.30 AM – 2.30 PM
GlycINATOR®, FabRICATOR® and GingisKHAN®
Wednesday, June 5
Analysis of O-glycosylated Biopharmaceuticals using an O-glycan dependent Endoprotease and LC-MS
Andreas Nägeli
WP 334, Poster Session 10.30 AM – 2.30 PM
MALDI-in-source decay FT-ICR MS for top-down and middle-down characterization of monoclonal antibodies
Simone Nicolardi
WP 032, Poster Session 10.30 AM – 2.30 PM
Application of a label-free and domain-specific free thiol method in monoclonal antibody characterization
Yi Pu
WP 040, Poster Session 10.30 AM – 2.30 PM
Collision induced unfolding experiments to decipher the structural regions of a hybrid monoclonal antibody
Thomas Botzanowski
WP 481, Poster Session 10.30 AM – 2.30 PM
Thursday, June 6
Intact and Subunit Mass Analysis Using Native Ion Exchange Chromatography Coupled to an Orbitrap Mass Spectrometer
Qian Liu
ThP 663, Poster Session 10.30 AM – 2.30 PM
Ultra-Fast Analysis of Intact Proteins Using SPE- TOF
Kevin McCann
ThP 149, Poster Session 10.30 AM – 2.30 PM
Isomeric linkage determination of Sialic acid on O-glycopeptides using O-protease and LC-MS/MS
Jieqiang Zhong
ThP 071, Poster Session 10.30 AM – 2.30 PM
Poster Presentations at PEGS Europe 2018
This week, scientists from Genovis are presenting two poster at the Protein Engineering Summit in Lisbon, Portugal. The posters cover our O-glycan specific endoprotease Operator and the recently launched FabRICATOR-HPLC column for automated antibidy subunit generation. Check out the poster abstracts below:
An O-glycan Specific Endoprotease with Applications in Glycoprotein Analysis using LC-MS
Helen Nyhlen, Maria Nordgren, Stephan Björk, Rolf Lood, Fredrik Leo, Fredrik Olsson
Genovis AB, Sweden
Changes in protein glycosylation may have an impact on the structure and function of a glycoprotein and O-glycosylation has drawn more and more attention for its roles in a wide range of biological processes. Characterization of glycosylation is of growing importance for the development and quality control of recombinant glycoprotein drugs and biosimilars. The study of O-linked glycosylation within the field of glycoproteomics is however challenging due to complicated sample preparation, difficult analytical procedures and the lack of O-glycan specific enzymes.
An O-glycan specific protease originating from the mucin degrading bacteria Akkermansia muciniphila has been described previously. The enzyme is dependent on the presence of O-glycans for digestion and hydrolyzes the peptide bond N-terminally to O-glycosylated serine and threonine residues. This feature can be used for the generation of intact O-glycopeptides to study site occupancy and composition of O-glycans in various biologic samples. We present here workflows that enabled determination of O-glycan sites and composition for O-glycosylated biopharmaceuticals and for proteins in human serum.
The O-linked glycosylation sites of biopharmaceuticals were assessed by treatment with PNGaseF, sialidases, O-protease and/or trypsin overnight prior LC/MS. The unique MS/MS peptides obtained revealed and defined the O-glycosylated threonine and serine residues. Enrichment of O-glycoproteins from human serum was achieved in native conditions using an affinity binding resin for O-glycan protein based on agarose beads with immobilized inactive O-protease. The complex protein sample was desialylated during the incubation step for binding. Bound proteins were then eluted by urea and treated with PNGaseF, active O-protease and/or trypsin followed by RP-C18 or HILIC separation and ESI-QTOF/MS analysis. The resin displayed high affinity for core 1 mucin-type glycans. With this workflow peptides and O-glycopeptides, with site-specific information, from several serum proteins were identified.
To summarize, using the characteristics of the O-protease and the O-glycoprotein affinity binding resin, strategies for the characterization of O-glycosylated proteins from pure and complex protein samples have been developed. The O-protease and the O-glycoprotein binding resin are potentially useful tools for deep characterization of O-glycoproteins.
Rapid On-column Digestion for Automated Monoclonal Antibody Analysis
Stephan Björk, Andreas Nägeli, Maria Nordgren, Linda Andersson, Helen Nyhlen, Jonathan Sjögren, Fredrik Olsson
Genovis AB, Lund, Sweden
Monoclonal antibodies (mAbs) and other IgG-based biopharmaceuticals are a fast-growing market. The inherent heterogeneity of such biologics necessitates detailed characterization by liquid chromatography and mass spectrometry (LC-MS) during development and production. While bottom-up peptide mapping is still the gold standard for analysis of critical quality attributes, such approaches are resource and time intensive in terms of both data acquisition and analysis. Top-down and middle-down approaches are therefore gaining in popularity. Antibody subunit analysis has become a widely accepted analytical strategy for rapid characterization of therapeutic antibodies and related products. The IdeS enzyme specifically digests IgG just below the hinge, generating F(ab’)2 and Fc/2 fragments. Reduction of disulfide bonds yields fragments of 23-25kDa in size which are amenable to high-resolution mass spectrometry. The IdeS based middle-level LC-MS workflow therefore enables the analysis of multiple antibody quality attributes such as glycosylation, oxidation, and C-terminal lysine clipping.
Here we present a rapid and automatable solution for antibody subunit generation in an HPLC column format. FabRICATOR (IdeS) enzyme was immobilized on the column to allow for automated middle-level analysis in a 2D-HPLC setup. The mAbs are digested on-column in the first dimension and the resulting subunits are separated and analyzed in the second dimension by RP-HPLC. This could be achieved with minor modifications to an HPLC-MS setup and potentially be connected directly to a bioreactor for automated monitoring of an on-going mAb production. The column tolerates continuous operation at 37°C for >10 days without a significant decrease in digestion performance and delivers consistent results for Fc glycan analysis during the entire period of operation. Additionally, other critical quality attributes such as Fab glycosylation and lysine clipping could be monitored. FabRICATOR-HPLC provides a fast solution for antibody subunit generation while reducing sample handling errors and increasing throughput.